PL191812B1 - Sugar beet vegetable material consisting of mutant cells, method for producing immunity against herbicide in the sugar beet, method for obtaining immunity against herbicide in the sugar beet, method for controlling the weeds growing together with sugar be - Google Patents

Abstract

Sugar beet plants which are resistant to both imidazolinone and sulfonylurea herbicides are described. The sugar beet plants are derived from susceptible cells by sequential selection of SU-R cells, plant regeneration, and re-selection for IM-R. The resistant sugar beet plants derived from the cells can be grown in fields where imidazolinones and sulfonylureas have been used for weed control.

Description

The present invention relates to sugar beet plant material consisting of mutant cells, a method of producing herbicide resistance in sugar beet, a method of obtaining herbicide resistance in beet, a method of controlling weeds growing together with sugar beet.

According to the invention, it is possible to produce sugar beet (Beta vulgaris L.) which is resistant to both the imidazolinone herbicides and the sulfonylurea herbicides used to control weeds. The method of producing herbicide resistance in sugar beet according to the invention uses a mutation of the gene encoding acetolactate synthase (ALS), also known as acetohydroxy acid synthase (AHAS), by applying herbicides sequentially to cells in the culture medium.

A genetic alteration of the acetolactate synthase gene has previously been described using genetic engineering methods as shown in US Patent Nos. 5,013,659; 5141870 and 5378824. This type of modification of sugar beet is shown in Example IV of Patent 5378824. These results were less than satisfactory in producing plants that reproduced imparting herbicide resistance.

Saunders et al. In Crop Science 32: 1357-1360 (1992) also describe the production of sugar beet (CR1-B), which is resistant to sulfonylureas, from a susceptible, self-pollinating clone (REL-1) by selecting mutant cells in herbicide-containing culture medium. Various resistant plants have been produced and crossed. Hart et al. (Weed Sci. 40: 378-383 (1992); and Weed Sci. 41: 317-324 (1993)) further characterized the resistant line and concluded that the resistance was due to altered ALS activity and showed no cross-resistance against other herbicides that inhibit ALS and that it was encoded by a single, partially dominant gene.

There are no publications describing imidazolinone resistance obtained by modifying ALS in sugar beet. There are no literature descriptions of plants that are resistant to both imidazolinone and sulfonylurea herbicides. Various maize lines with imidazolinone resistance have been obtained as described by Newhouse et al., Theotetical and Applied Genetics 83: 65-70 (1991).

A commercial method of crop protection is the use of clinical "safeners, such as those described in European Patent No. 375875. This method introduces another chemical into the soil. A preferred method is to obtain sugar beet that is resistant to the imidazolinone and sulfonylurea herbicides.

It is therefore an object of the present invention to provide a method for obtaining imidazolinone and sulfonylurea herbicide resistance in sugar beet by mutating the acetolactate synthase gene encoding such resistance. Moreover, it is an object of the present invention to provide sugar beet which is resistant to these herbicides. These and other purposes will become more apparent by reference to the following description and drawings.

Fig. 1 is a schematic representation of the method of the present invention for producing imidazolinone and sulfonylurea resistant sugar beet 93R30A or B. R = resistant; IM = imidazolinone; S = sensitive; SU = sulfonylurea; Sur = ALS allele dominant for SU-R, TP-R and IM-S; SB = ALS allele dominant for SU-R, TP-R and IM-R; TP = triazolopyrimidine. 93R30B is SU-R and IM-R and TP-R. 93R30A (but not 93R30B) is homozygous for the Sur allele (SU-R, IM-S). This was the starting material for imidazolinone resistance.

Fig. 2 is a diagram detailing the steps involved in producing the mutant sugar beet.

Fig. 3 is a graph showing the ALS assay for imazethapyr 93R30B resistance.

Fig. 4 is a graph showing the amino acid substitutions observed for wild-type (wt) three mutant sugar beet (Sir-13, Sur and SB) and the corresponding herbicide resistance. Only amino acid residues that have been reported in the literature for herbicide resistance are included. In the single letter amino acid code, A = alanine, P = proline, W = tryptophan, S = serine, and T = threonine. Boxed letters indicate changes to the wild-type sequence.

Figures 5A through 5D are photographs showing the response of a whole sugar beet plant to imazethapyr applied postemergence.

Figures 6A through 6D are photographs showing the response of a whole sugar beet plant to AC 299263 applied post-emergence.

PL 191 812 B1

Fig. 7 is a photograph showing ALS enzyme response to imazethapyr in vitro.

Fig. 8 is a photograph showing ALS enzyme response to flumetsulam in vitro.

Figures 9A through 9D are photographs showing the response of a whole sugar beet plant to chlorsulfuron applied post-emergence.

Fig. 10 is a photograph showing ALS enzyme response to chlorsulfuron in vitro.

The present invention relates to sugar beet plant material consisting of mutant cells with a mutant acetolactate synthase gene encoding a synthase, wherein the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337, wherein the mutant cells have resistance to both imidazolinone and sulfonylurea herbicides and resistance can be transferred by conventional crossing plants made from cells.

Preferably the material is a material obtained from sensitive cells of the parent sugar beet designated CR1-B, the seed of which has been deposited as ATCC 97961 and having resistance to sulfonylurea herbicides, but not having resistance to imidazolinone herbicides, by culturing the sensitive cells in an imidazolinone herbicide culture medium. to select mutant cells.

Preferably, the material is a seed or a material derived from a seed.

Furthermore, the present invention relates to a method for producing herbicide resistance in sugar beet which comprises:

a) treating with an imidazolinone herbicide in the culture medium of starting sugar beet cells with a mutant acetolactate synthase gene encoding a synthase, wherein the nucleotides are modified from cytosine to thymine at position 562, the starting cells being homozygous for resistance to sulfonylurea herbicide culture medium; and

b) selecting second cells having resistance to both herbicides, whereby the resistance may be transferred by crossing plants containing the second cells.

Preferably, the second cells were obtained from the starting sugar beet cells designated 93R30 (SurSur), deposited as ATCC 97961, by treating the 93R30 cells with an imidazolinone herbicide.

Preferably, the second cells are in the seed deposit under number ATCC 97535 (93R30B).

The present invention also relates to a method for obtaining herbicide resistance in beet which comprises:

a) treating the starting sugar beet cells with an imidazolinone herbicide in a culture medium, wherein the starting cells have a homozygous sulfonylurea herbicide resistance in the culture medium;

b) selecting second cells having resistance to both herbicides;

c) growing from the second cells a first plant that has herbicide resistance and a mutant acetolactate synthase gene encoding a synthase, wherein the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337; and

d) crossing the first plant with a second plant to produce a crossed plant which has herbicide resistance.

Preferably, in the method of the invention, the starting cells in step (a) are obtained from the sugar beet designated 93R30, deposited as ATCC 97961, by treating 93R30 cells with an imidazolinone herbicide.

Preferably also the second cells are in the seed deposit and are designated ATCC 97535 (93R30B).

Furthermore, the invention relates to a method for controlling weeds growing together with sugar beet, the method comprising:

a) Sowing a field with sugar beet seeds containing cells with a mutant acetolactate synthase gene encoding a synthase in which the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337, whereby the mutant cells have resistance to both imidazolinone and imidazolinone herbicides sulfonylureas, and resistance can be transferred by conventional crossing plants made from cells; and

b) applying an imidazolinone herbicide to a plant in the field for weed control.

PL 191 812 B1

Preferably the plant is derived from sensitive sugar beet parental cells designated CR1-B deposited as ATCC 97961 and having resistance to sulfonylurea herbicides, but not having resistance to imidazolinone herbicides, by culturing the sensitive cells in a culture medium with the imidazolinone herbicide mutated to select .

Preferably the plant is derived from seed deposited as ATCC 97535 (93R30B).

REL-1 sugar beet (Regenerating, East Lansing-1) is available from Dr. Joseph Saunders, Genetics, US Department of Agriculture, East Lansing, Michigan, and was disclosed in 1987. It is available free of charge. REL-1 is sensitive (S) to the herbicides imidazolinone (IM-S), sulfonylurea (SU-S) and triazolopyrimidine sulfonanilide (TP-S) as shown in Figure 1.

Using callus and suspension cultures, a line was obtained that was heterozygous for imidazolinone and sulfonylurea resistance (IM-R and SU-R). This cell line (seed) was deposited with the American Type Culture Collection as ATCC 97535 on May 6, 1996 under the Budapest Treaty and is referred to herein as 93R30B. The cell line is available upon request by name and number. By crossing with better lines of sugar beet, in particular Beta vulgaris L, a resistant beet which is extremely commercially useful can be produced.

Sugar beet resistant to imidazolinone herbicides, in particular imazethapyr and sulfonylurea (chlorsulfuron) herbicides solve the problem of soil residues after application of this herbicide in sugar beet growing areas. Currently, there is a rest period of 40 months between the application of such a herbicide and the use of such a field for growing sugar beet. Furthermore, the high level of imidazolinone and sulfonylurea resistance enables the use of imidazolinone herbicides for weed control in sugar beet.

The following examples show the isolation and crossing of the new sugar beet 93R30B as illustrated in Fig. 1.

P r z y k ł a d l

1. Selection of the sugar beet variety, 93R30B, resistant to imidazolinone and sulfonylurea

Description of the material. Rel-1 (Regenerating East Lansing-1) is a highly regenerative male fertile sugar beet line used for the preselection of mutant cells resistant to sulfonylurea herbicides and used as a sensitive control line in most experiments.

CR1-B. This sugar beet isolate was produced by culturing Rel-1 cells in chlorsulfuron-containing medium. This variant is sulfonylurea resistant, has little resistance to the triazolopyrimidine herbicide, flumetsulam, but is not resistant to imidazolinone. It has been reported by Saunders et al. (Crop Sci. 32: 1357-1360) and Hart et al. (Weed Sci. 40: 378-383 and 41: 317-324). The SU resistance gene was named Sur. 93R30B

This isolate was produced by culturing the highly regenerative cells, a Sur homozygous sugar beet plant (derived from CR1-B), on medium containing imazethapyr.

EL-49 East Lansing 49, disclosed by the USDA in 1993.

Homozygous for the Sur gene sugar beet line used as a control for sulfonylurea resistance and imazethapyr sensitivity.

a. Plants were selected as starting material for somaclonal selection based on their ability to produce callus in the presence of high concentrations of cytokinins (in B1 medium) or to regenerate callus shoots.

Recipe for medium B1:

gl ^{-1 of} sucrose

100 mg l ^-1 myo-inositol

1.65 g1 ^{-1 NH4NO3}

1, 90 gl ^-1 KNO3

0.44 g1 ^-1 CaCl2 x 2H2O

0.37 g ^-1 MgSO4 x 7H2O

0.17 g ^-1 KH2PO4

6.2 mg l ^-1 H3BO3

16.8 mg l ^-1 MnSO4 x H2O

PL 191 812 B1

10.6 mg l ^-1 ZnSO4 x 7H2O

0.88 mg l ^-1 KI

0.25 mg l ^-1 Na2MoO4 x 2H2O

0.025 mg l ^-1 CuSO4 x 5H2O

0.025 mg l ^-1 CoCl2 x 6H2O

37.3 mg l ^{-1 Na2EDTA}

27.8 mg l ^-1 FeSO4 x 7H2O mg l ^-1 thiamine

0.5 mg l ^-1 pyridoxine

0.5 mg l ^-1 nicotinic acid mg l-1 benzylaminopurine pH 5.95.

Solid B1 medium was autoclaved with 9 gL ^-1 plant culture agar and supplemented with filter-sterilized basic herbicide solution as required by the selection scheme. The agar-containing medium was poured onto 15 x 100 mm plastic petri dishes.

The liquid medium B1 was dispensed 40 ml into 125 ml Erlenmeyer flasks and autoclaved for use in the cultivation of the liquid suspensions.

The starting material for the selection of the imidazolinone resistant variant, 93R30B, was 93R30 (Sur homozygous, derived from CR1-B).

In details:

1. Cells from Rel-1 (SU-S, IM-S, TP-S) were placed in cultures that selected them for their ability to grow on chlorsulfuron and the plant was regenerated, CR1-B (described by Saunders et al. Crop Sci. 32 : 1357-1360 (1992)).

2. CR1-B was crossed with 293 (a more typical beet), the F1 generation was selfed, and plants homozygous for SU-R, IM-S and TP-R were identified. The plant, known as EL-49, with the same characteristics was disclosed in 1993 by Saunders (the gene was named Sur and the plant was homozygous for the gene).

3. Later, homozygotes for the Sur gene derived from CR1-B were selected for their ability to regenerate plants from culture. They were used as starting material (e.g. 93R30) in culture for selection for imazethapyr.

4. The isolates known as 93R30A and 93R30B and the regenerated plants were derived from the above selection. These have the properties of CR1-B and IM-R. The gene responsible for IM-R appears to be an altered form of Sur and gives identity to SB.

Leaf disc explants were produced from rapidly expanding leaves of the starting plant (93R30). The leaf surface was sterilized (step 1) for two consecutive 20 min. rinses with 15% commercial bleach with 0.025% Triton X-100 (T-9284, Sigma Chemical Co., St. Louis, MO). Leaves were rinsed twice with sterile deionized water. Leaf discs were cut using a flame sterilized # 3 cork.

The leaf discs were placed sterile on solid B1 medium (step 2). The discs were incubated in the dark at 30 ° C for 4-8 weeks until brittle, white callus tissue had grown out of the leaf disc. The callus tissue was mechanically separated with forceps and then transferred to 125 ml Erlenmeyer flasks (step 3) containing 40 ml of liquid B1 medium. The flasks were placed on a rotary shaker at 50 Hz under low intensity fluorescent (30 µ / m2). After 1 week, the liquid cultures were transferred to fresh B1 medium.

Two weeks after starting the liquid culture, the cell flocs were separated using a cell separator (Sigma CD-1) with a density of 60 (60 mesh). After the cells were pelleted, approximately two-thirds of the volume of the liquid medium was removed.

In step 4, cells were resuspended in leftover media and 1 ml aliquots were spread evenly over solid B1 media supplemented with 50 nM the herbicide imazethapyr (PURSUIT, American Cyanamid, Wayne, NJ). Plates were wrapped and incubated under fluorescent light (5-10 µM / rms) for 8-12 weeks.

b. At 50 nM imazethapyr concentration, all sensitive cells died. Cell clots that survived and grew at this concentration were identified as possible resistant variants. In step 5, the cell flocs (1-3 mm in diameter) were transferred to fresh, solid B1 medium, without herbicide, and allowed to grow under low light (10-20 µ / m2). Each putative resistant variant was individually identified and grown separately.

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c. In step 6, the white, brittle callus was transferred every 4-6 weeks to fresh solid B1 medium until single shoots (or shoot) had grown out of it. These shoots were transferred to shoot growth medium (M20).

Recipe for M20 Medium:

gl ^{-1 of} sucrose

100 mg l ^-1 myo-inositol

1.65 g ^-1 NH4NO3

1.90 gl ^-1 KNO3

0.44 g1 ^-1 CaCl2 x 2H2O

0.37 g ^-1 MgSO4 x 7H2O

0.17 g ^-1 KH2PO4

6.2 mg l ^-1 H3BO3

16.8 mg l ^-1 MnSO4 x H2O

10.6 mg l ^-1 ZnSO4 x 7H2O

0.88 mg l ^-1 KI

0.25 mg l ^-1 Na2MoO4 x 2H2O

0.025 mg l ^-1 CuSO4 x 5H2O

0.025 mg l ^-1 CoCl2 x 6H2O

37.3 mg l ^-1 Na2EDTA

27.8 mg l ^-1 FeSO4 x 7H2O mg l ^-1 thiamine

0.5 mg l ^-1 pyridoxine

0.5 mg l ^-1 nicotinic acid

0.25 mg l ^-1 benzylaminopurine pH 5.95

Solid M20 medium was autoclaved with 9 gL ^-1 plant culture agar and supplemented with filter-sterilized stock herbicide as required by the selection scheme. The agar-containing medium was poured onto 20 x 100 mm plastic petri dishes.

Shoot cultures were grown under fluorescent illumination at 10-20 µ / m < 2 > and propagated by cutting the shoot culture with a scalpel blade. Selected shoot cultures were propagated and their resistance levels assessed as described below.

2. Level of resistance to imidazolinone, sulfonylurea and triazopyrimidine sulfanilide of shoot culture.

In step 7, the herbicide resistance of the shoot culture was assessed by placing three small slices of shoots of similar size in each of the dishes containing M20 medium supplemented with logarithmically increasing concentrations of imidazolinone herbicide, sulfonylurea, or triazopyrimidine sulfonanilide, ranging from 1 nM to 0.1 mM. . The 93R30 culture responses were compared with shoot cultures of a sensitive control (Rel-1) and a chlorsulfuron resistant and imidazolinone sensitive control (EL-49). The shoots were cultured at a light intensity of 20 µ / m 3 and 25 ° C for 3 weeks. Shoot culture response was determined by assessing shoot damage and fresh weight, 3 weeks after transfer to selection medium. The amount of herbicide resistance was determined by the ratio of the resistance value to the sensitivity I50 (concentration of the herbicide causing 50 percent damage).

The cross-resistance characteristics of each variety were determined as above by changing the herbicide used in the medium. Shoot culture 93R30B showed 3,600-fold resistance to imazethapyr, 10,000-fold resistance to chlorsulfuron, and showed 60-fold cross-resistance to the triazolopyrimidine sulfonanilide herbicide, flumetsulam, as shown in Table 1. For comparison, only 3-fold EL-49 show levels. imazethapyr resistance.

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T a b e l a 1

Shoot culture resistance size and cross resistance

Herbicide Herbicide family ⁶ Rel-1 93R30B EL-49 I50 ⁵ (pM) I50 (pM) R / S ⁴ I50 (pM) R / S Imazethapyr THEM 0.018 64 3600X 0.053 3X Imazametabenzomethyl THEM 0.80 > 100 > 125X twenty 25X Imazakwin THEM 0.040 16 400X 0.040 1X AC299 / 263 ¹ THEM 0.030 40 1400X 0.070 3X Chlorosulfuron ² SU 0.0025 25 10000X 25 10,000Χ Flumetsulam ³ TP 0.028 1.7 60X 1.1 40X

¹ - Imidazolinone RAPTOR (American Cyanamid, Wayne, NJ) ² - Sulfonylurea - Glean (Dupont, Wilmington, DE) ³ - Triazolopyrimidine sulfanilide (BROADSTRIKE, Dow Elanco, Indianapolis, IN).

⁴ - R / S = ratio of resistance to sensitivity values of the control I50 for the specific herbicide tested ⁵ - I50 is the herbicide concentration required to induce 50% damage in shoot culture ⁶ - Herbicide family classification: IM, imidazolinone; SU, sulfonylurea, and TP, a triazolopyrimidine sulfonanilide

3. Plant regeneration and breeding

In step 8, the stem cultures of 93R30B were regenerated into whole plants in a similar manner. Two weeks after the first transfer of the culture, 93R30B shoots were transferred to 125 ml Erlenmeyer flasks containing 40 ml N3 medium to allow root formation.

Recipe for N3 Medium:

gl ^{-1 of} sucrose

100 mg l ^-1 myo-inositol

1.65 g1 ^{-1 NH4NO3}

1, 90 gl ^-1 KNO3

0.44 g1 ^-1 CaCl2 x 2H2O

0.37 g ^-1 MgSO4 x 7H2O

0.17 g ^-1 KH2PO4

6.2 mg l ^-1 H3BO3

16.8 mg l ^-1 MnSO4 x H2O

10.6 mg l ^-1 ZnSO4 x 7H2O

0.88 mg l ^-1 KI

0.25 mg l ^-1 Na2MoO4 x 2H2O

0.025 mg l ^-1 CuSO4 x 5H2O

0.025 mg l ^-1 CoCl2 x 6H2O

37.3 mg l ^{-1 Na2EDTA}

27.8 mg l ^-1 FeSO4 x 7H2O mg l ^-1 thiamine

0.5 mg l ^-1 pyridoxine -1

0.5 mg l ^-1 nicotinic acid mg l ^-1 naphthalene acetic acid pH 5.95

To each 125 ml Erlenmeyer flask containing 40 ml of N3 medium, 9 gL ^{-1 of} agar (0.45 g) was added and the medium was autoclaved for use in rooting permeable culture.

The cultures were then placed in daylight for 24 h with average light intensity (40-60 µ / m2) at 25 ° C. Roots generally formed 6-8 weeks later.

In step 9, the rooted shoots (R0 generation) were transferred to Baccto potting medium (Michigan Peat Co., Huston, TX) in a greenhouse. In step 10, R0 plants from 93R30B regenerates were crossed with a smooth root sugar beet named 293 or with REL-1. F1 seeds from these crosses were self-pollinated to obtain F2 seeds.

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Imidazolinone resistance was assumed to be a dominant or semi-dominant monogenic trait. The F2 generation, formed by self-pollination of imidazolinone-resistant and sulfonylurea-resistant F1 plants, should segregate in a ratio of 1 homozygous resistant: 2 heterozygous resistant: 1 homozygous sensitive to both imidazolinone and sulfonylurea.

Evidence suggests that imidazolinone resistance is the dominant trait in 93R30B. This conclusion is derived from the analysis of the F1 progeny, from the cross between 93R30B and herbicide sensitive, male fertile 293 or Rel-1 sugar beet lines that segregate 1: 1 for IM-R or IM-S. Moreover, in all the F2 and backcrosses (BC1) of the plants tested so far, IM-R and SU-R segregate together. This suggests that the two resistances are linked to each other and correspond to independent changes in the same ALS allele. The resistance or sensitivity of F1 plants to imazethapyr and chlorsulfuron was determined using a non-damaging leaf disk outgrowth test.

Test protocol:

This protocol is a non-plant-damaging test designed to early screen beet segregation for herbicide resistance in order to establish its resistant or susceptible status without having to spray the beets after they have grown in a greenhouse.

1. The newly developed leaf (at least the fourth true leaf) was trimmed from the plant.

2. The leaf surface was sterilized for two 20 min. rinses with 15% commercial bleach with 0.025% Triton X-100, followed by rinsing twice with sterile deionized water.

3. Leaf disc explants were cut using a flame sterilized # 3 cork.

4. Leaf disks were transferred to herbicide-free B1 medium containing 100 nM imazethapyr or 100 nM chlorsulfuron. The dishes were divided into four sectors so that four samples could be tested on a single dish. Four to five leaf discs were placed on each plate for each sample.

5. The dishes were wrapped and incubated at 25 ° C for 4-7 days under low light (approximately 10 µM / rms).

6. Resistant samples were identified by their viability and growth in selection medium. Sensitive samples did not expand and turned yellow to brown. The results were compared with the growth of the leaf discs placed on a medium without the herbicide to confirm the accurate determination of the expected level of disc expansion.

4. ALS Target Enzyme Imazethapyr Response

Standard procedures were used to partially purify ALS from rapidly growing leaves grown in a greenhouse of sugar beet plants (Hart et al., Weed Science 40: 378-383 (1992)). Extracts from heterozygous plants of the F1 93R30B generation and S1 Rel-1 seeds were tested for ALS activity in the presence of logarithmically increasing concentrations of imazethapyr. ALS activity was determined for leaf extracts of the sugar beet lines REl-1 and 93R30B. Plants were grown in greenhouses as described above to the fourth to sixth leaf stage. ALS activity from fresh extracts was determined in the presence of imazethapir and chlorsulfuron at logarithmically increasing concentrations. The methods and procedures were modified from those reported by Ray (Plant Physiol. 75: 827-831 (1984)) and Shaner et al. (Plant Physiol. 76: 545-546 (1984)). Ten grams of sugar beet leaves were homogenized in 40 ml of cold homogenization buffer (0.1 MK ₂ HPO ₄ ), pH 7.5, 1 mM sodium pyruvate, 0.5 mM MgCl ₂ , 0.5 mM thiamine pyrophosphate, 10 μΜ dinucleotide flavin adenine 10% v / v glycerol) with 2.5 g polyvinylpyrrolidone. The homogenate was filtered through eight layers of gauze and centrifuged at 27,000 g for 20 min. The supernatant was collected and brought to 50% saturation with (NH4) 2SO4. This saturated solution was kept at 0 ° C for 1 h, then centrifuged at 18,000 g for 15 min., The pellet was resuspended in 1 ml of suspension buffer (0.1 M K2HPO4, pH 7.5, 20 mM sodium pyruvate, 0, 5 mM MgCl2) and desalted on a Sephadex G-25 PD-10 ⁵ column. The enzyme extracts were immediately tested.

ALS activity was measured by adding 0.2 ml of enzyme preparation (diluted 3: 1 in suspension buffer) to 1.3 ml of reaction buffer (25 mM K2HPO4, pH 7.0, 0.625 mM MgCl2, 25 mM sodium pyruvate, 0.625 mM thiamine pyrophosphate, 1.25 μΜ flavin adenine dinucleotide and incubated at 35 ° C for 1 hour The reaction mixture contained a final concentration of 0, 4, 40, 400, 4,000, 40,000, 400,000 or 4,000,000 nM imazethapyr or 0, 0.9, 9, 90, 900, 9000, 90,000 nM of chlorsulfuron The reaction was stopped by adding 50 µl of 6N H ₂ SO ₄ and incubating at 60 ° C for 15 min This procedure as described by Westerfield (J. Biol. Chem. 16: 495-502 ( 1945)) also allows the decarboxylation of the ALS enzyme product, acetolactate, to form acetoin.

The acetoin complex is prepared by adding 1 ml of 2.5% by weight α-naphthol and 0.25% by weight creatine in 2.5 N NaOH and incubating at 60 ° C for 15 min.

Commercial acetoin was used as a standard in the colorimetric reaction. The acetoin concentrations were determined by measuring the absorption of the reaction mixture at 530 nm. Experiments were carried out in triplicate for each herbicide. Protein concentrations in the extracts were determined by the method of Bradford (Anal. Biochem. 72: 248-254 (1976)) using bovine serum albumin for the standard curve.

The experiments were carried out twice and with triplicate replications. Fig. 3 shows the responses of the ALS extracts from 93R30B homozygous and sensitive Rel-1. 93R30B shows, at the enzyme level, a 1750 fold level of resistance to imazethapyr (as determined by the ratio of I50 values for resistant and susceptible lines).

5. Number of ALS gene copies

Southern analysis was performed to determine the number of gene copies present in sensitive sugar beet. Genomic DNA was isolated from sensitive F3 plants derived from the starting Sir-13 mutant and analyzed using commonly used techniques (Current Protocols in Molecular Biology, J. Wiley & Sons, New York (1991)). Similarly, genomic DNA from a commercial sugar beet variety was analyzed. A single copy of the ALS gene was detected in both sugar beet lines. These data would indicate that all ALS enzyme activity derived from the homozygous sugar beet mutant type would consist of the resistant form of the enzyme. This also supports the assumption that the SB change observed in isolate 93R30B is an altered form of ALS of the Sur allele. This was confirmed as described below.

6. Mutation in the ALS gene

To establish the molecular basis of ALS enzyme resistance, standard techniques were used to sequence the two regions of the ALS gene where all the herbicide resistance mutations previously reported (Current Protocols in Molecular Biology, J. Wiley & Sons, New York (1991)) were present. The sugar beet ALS gene was previously sequenced (US Patent No. 5,378,824) and Polymerase Chain Reaction (PCR) primers were designed to amplify the two regions of this gene responsible for the previously described herbicide resistance cases in plants. Comparison of the obtained sequences from sugar beet SU-R IM-S TP-R (Sur), SU-R IM-R TP-R (SB) and sensitive (Rel-1) showed that the herbicide resistance feature of the 93R30B line is caused by two independent mutations in the ALS gene.

The SU-R IM-S TP-R (Sur) inducing mutation is the result of a single nucleotide change from cysteine to thymine at position 562 in the nucleotide sequence resulting in a proline to serine substitution at position 188 of the sugar beet ALS amino acid sequence. This site has been associated with sulfonylurea resistance in Arabidopsis thaliana (Haughn et al., Molecular and General Genetics 211: 266-271 (1988), tobacco (Lee et al., EMBO J. 7: 1241-1248 (1988) and yeast (US Patent No. 5,378,824) No other base changes were observed from the wild-type nucleotide sequence in the two regions of the ATS gene tested.

The mutation causing SU-R IM-R TP-R (SB) is the result of two independent mutations in the ALS nucleotide sequence resulting in a change of two different amino acids. The first change is identical to the Sur mutation described above. This was expected since a sugar beet plant (93R30) was used as the starting material for the IM-R selection. In addition to this point mutation, an additional single nucleotide change from guanine to adenine at position 337 was observed. This second mutation resulted in the substitution of alanine for threonine at position 113 in the amino acid sequence of sugar beet ALS. This site has previously been reported to be associated with imidazolinone resistance in burdock (Bernasconi et al., J. Biol. Chem. 270: 17381-17385 (1995)) and sulfonylurea resistance in yeast (US Patent No. 5,378,824). No other base changes were observed from the wild-type nucleotide sequence in both regions of the ATS gene tested.

The following information shows the synergism of herbicide resistance possible mutations Ala ₁₁₃ --Thr and Pro _{188 -} Ser

The SB sugar beet ALS gene, a double mutant, provides herbicide resistance, which is a combination of families of chemicals that each single sugar beet mutant separately (ie, Sur or Sir-13) has. Fig. 4 shows the cross resistance to basic families characteristic of the two single mutants (Sur and Sir-13) and the double mutant (SB). The amino acids shown in Fig. 4 show 5 strictly conserved residues described in the literature,

PL 191 812 B1 as involved in resistance to an ALS inhibitory herbicide in plants. The interaction of independent mutations in the enzyme of the double mutant (and the corresponding plants) is unique. The combination of IM specific resistance observed in the Sir-13 amino acid substitution (Ala ₁₁₃ - -Thr) and SU and TP resistance observed in the Sur (Pro ₁₈₈ -Ser) substitution gives sugar beet 93R30B (aka SB) resistance to all three chemical classes herbicides.

However, resistance is not additive. Instead, the mutations are synergistic with respect to resistance to the imidazolinone herbicides imazethapyr and AC 299263. This phenomenon can be seen in Figures 5 and 6 and quantified in Table 2.

T a b e l a 2

Whole sugar beet response to postemergence ALS inhibitory herbicides

Sugar beet line Rel-1 Sir-13 Sur 93R30B Herbicide Grade I50 (g ha ^-1 ) R / S R / S R / S Imazethapyr IMI 0.5 100X 3X 300X AC299, 263 IMI 1.1 130X 4X > 250X Chlorosulfuron SU 0.07 1X > 1000X 20X Sulfometuron SU 0.2 1X 23X 4X

A single mutation in sugar beet, Sur and Sir-13, shows 3 and 100 fold resistance to imazethapyr; whereas the SB double mutant shows 300-fold resistance to the herbicide when applied post-emergence to whole plants. Confirmation of this synergism is provided by the observed ALS enzyme responses from each mutant to imazethapyr (Fig. 7). Moreover, the herbicide resistance in the SB double mutant (> 1000X) versus the single mutants, Sur (1X) and Si-13 (40X) is synergistic.

A similar synergism is apparent for the TP herbicide class. The shoot resistance of the Sur and Sir-13 single mutants is 40X and 1X, respectively, and 60X for the double mutant, SB. Data on the ALS enzyme confirm the observation of synergism with 50X and 3X flumetsulam resistance in the single mutants of Sur and Sir-13, respectively, and 200X for the double mutant, SB (Fig. 8). The second mutation observed in SB actually lowers the resistance to chlorsulfuron (a class of SU herbicides) observed at the whole plant level (Fig. 9). For chlorsulfuron, whole plant resistance of the SB double mutant (20X) is reduced more than 50-fold by adding the equivalent of the Sir-13 mutation (Ala113-Thr) (1X resistance) to the SU resistance gene, Sur (Pro188-Ser) (> 1000X resistance) (Tab. 2). The cause of antagonism between a single Sur mutant and a double SB mutant is not yet known. At the ALS enzyme level, synergistic resistance is also observed with chlorsulfuron (Fig. 10).

It should be considered that selection for imidazolinone resistance should be done first, followed by selection for chlorsulfuron resistance.

The above description is merely illustrative of the present invention and the present invention is limited by the claims below.

The amino acid sequences in Figure 4 are shown as SEQ ID NOS: 1, 2, 3, and 4.

Claims (12)

CLAIMS 1. Sugar beet plant material characterized in that it consists of mutant cells with a mutant acetolactate synthase gene encoding a synthase in which the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337 wherein the mutant cells have resistance to both imidazolinone and sulfonylurea herbicides and resistance can be transferred by conventional crossing plants generated from cells. 2. The material according to claim 1 which has been obtained from sensitive cells of the parent sugar beet designated CR1-B, the seed of which has been deposited as ATCC 97961 and having resistance to sulfonylurea herbicides, but not having resistance to imidazolinone herbicides, by culturing the sensitive cells in a culture medium with an imidazolinone herbicide to select mutant cells. Plant material according to claim 1 1, as seed or material derived from seed. 4. A method of producing herbicide resistance in sugar beet comprising: a) treating with an imidazolinone herbicide in the culture medium of starting sugar beet cells with a mutant acetolactate synthase gene encoding a synthase, wherein the nucleotides are modified from cytosine to thymine at position 562, the starting cells being homozygous for resistance to sulfonylurea herbicide culture medium; and b) selecting second cells having resistance to both herbicides, whereby the resistance may be transferred by crossing plants containing the second cells. 5. The method according to p. The process of claim 4, wherein the second cells were obtained from the starting sugar beet cells designated 93R30 (SurSur), deposited as ATCC 97961, by treating the 93R30 cells with an imidazolinone herbicide. 6. The method according to p. The method of claim 4, wherein the second cells are on the seed deposit under number ATCC 97535 (93R30B). 7. A method for obtaining herbicide resistance in beet, characterized by comprising: a) treating the starting sugar beet cells with an imidazolinone herbicide in a culture medium, wherein the starting cells have a homozygous sulfonylurea herbicide resistance in the culture medium; b) selecting second cells having resistance to both herbicides; c) growing from the second cells a first plant that has herbicide resistance and a mutant acetolactate synthase gene encoding a synthase, wherein the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337; and d) crossing the first plant with a second plant to produce a crossed plant which has herbicide resistance. 8. The method according to p. The method of claim 7, wherein the starting cells in step (a) are obtained from the sugar beet designated 93R30, deposited as ATCC 97961, by treating 93R30 cells with an imidazolinone herbicide. 9. The method according to p. The method of claim 7, wherein the second cells are in the seed deposit and are designated ATCC 97535 (93R30B). 10. A method of controlling weeds grown together with sugar beet, the method comprising: (a) Sowing a field with sugar beet seeds containing cells with a mutant acetolactate synthase gene encoding a synthase in which the nucleotides are modified from cytosine to thymine at position 562 and from guanine to adenine at position 337, wherein the mutant cells have resistance to both imidazolinone herbicides and and sulfonylureas, and resistance can be transferred by conventional crossing plants made from cells; and (b) applying an imidazolinone herbicide to a plant in the field to control weeds. 11. The method according to p. The method of claim 10, characterized in that the plant is derived from sensitive cells of the parent sugar beet designated CR1-B, deposited as ATCC 97961 and having sulfonylurea herbicide resistance but no herbicide resistance Imidazolinone compounds, by culturing the sensitive cells in a culture medium with an imidazolinone herbicide to select for mutant cells. 12. The method according to p. The process of claim 10, wherein the plant is derived from seed deposited as ATCC 97535 (93R30B).